New insight on the enteric cholinergic innervation of the pig colon by central and peripheral nervous systems: reduction by repeated loperamide administration.

Introduction: The central and peripheral nervous systems provide cholinergic innervation in the colon. The ability to assess their neuroanatomical distinctions is still a challenge. The pig is regarded as a relevant translational model due to the close similarity of its enteric nervous system (ENS) with that of human. Opioid-induced constipation is one of the most common side effects of opioid therapy. Methods: We developed an approach to differentiate the central and peripheral cholinergic innervation of the pig colon using double immunolabeling with a novel mouse anti-human peripheral type of choline acetyltransferase (hpChAT) antibody combined with a rabbit anti-common type of ChAT (cChAT) antibody, a reliable marker of cholinergic neurons in the central nervous system. We examined their spatial configurations in 3D images of the ENS generated from CLARITY-cleared colonic segments. The density was quantitated computationally using Imaris 9.7. We assessed changes in the distal colon induced by daily oral treatment for 4 weeks with the µ opioid receptor agonist, loperamide (0.4 or 3 mg/kg). Results: The double labeling showed strong cChAT immunoreactive (ir) fibers in the cervical vagus nerve and neuronal somata and fibers in the ventral horn of the sacral (S2) cord while hpChAT immunoreactivity was visualized only in the ENS but not in the vagus or sacral neural structures indicating the selectivity of these two antibodies. In the colonic myenteric plexus, dense hpChAT-ir neurons and fibers and varicose cChAT-ir fibers surrounding hpChAT-ir neurons were simultaneously visualized in 3D. The density of cChAT-ir varicose fibers in the outer submucosal plexus of both males and females were higher in the transverse and distal colon than in the proximal colon and in the myenteric plexus compared to the outer submucosal plexus and there was no cChAT innervation in the inner submucosal plexus. The density of hpChAT in the ENS showed no segmental or plexus differences in both sexes. Loperamide at the highest dose significantly decreased the density hpChAT-ir fibers + somata in the myenteric plexus of the distal colon. Discussion: These data showed the distinct density of central cholinergic innervation between myenteric and submucosal plexuses among colonic segments and the localization of cChAT-ir fibers around peripheral hpChAT neurons in 3D. The reduction of cholinergic myenteric innervation by chronic opiate treatment points to target altered prokinetic cholinergic pathway to counteract opiate constipation.

Experimental Study of Mechanical Integrity Testing in Stapled Large Bowel: Methylene Blue Leak Test Is Not Inferior to Air Leak Test.

BACKGROUND/OBJECTIVES: Anastomotic leakage remains the most devastating postoperative complication in colorectal surgery. The mechanical integrity of the newly formed colorectal anastomosis can be evaluated by visual inspection intraoperatively; both air leak and liquid leak tests are also used to evaluate the integrity of stapled colorectal anastomoses. It is not clear whether double-stapled anastomoses are more prone to leaks than single-stapled anastomoses. The aim of our study was to compare the methylene blue and the air leak test in the experimental setting of single-stapled and double-stapled porcine bowels. METHODS: Twenty-four distal colons were excised from slaughtered pigs without delay. The proximal bowel end was closed with a linear stapler using blue cartridges. The bowels were randomly divided into single-stapled or double-stapled groups. Air leak and methylene blue leak tests were performed. A digital pressure monitor with a gradual pressure increase function was used to both gradually increase pressure within the bowel and to determine the pressure at which the stapler line disintegrated. RESULTS: Air leakage occurred at a mean pressure of 51.62 (±16.60) mm Hg and methylene blue leakage occurred at 46.54 (±16.78) mm Hg (p = 0.31). The air and methylene blue leaks occurred at comparable pressures in single-stapled bowels and in double-stapled bowels (47.21 [±14.02] mm Hg vs. 50.96 [±19.15] mm Hg, p = 0.6). CONCLUSIONS: The methylene blue solution leak test is not inferior to the air leak test. There is no significant difference in bursting pressure between single-stapled and double-stapled anastomoses.

Preclinical evaluation of porcine colon resection using hollow core negative curvature fibre delivered ultrafast laser pulses.

Ultrashort pulse lasers offer great promise for tissue resection with exceptional precision and minimal thermal damage. Surgery in the bowel requires high precision and minimal necrotic tissue to avoid severe complications such as perforation. The deployment of ultrashort lasers in minimally invasive or endoscopic procedures has been hindered by the lack of suitable optical fibres for high peak powers. However, recent developments of hollow core microstructured fibres provide potential for delivery of such pulses throughout the body. In this study, analysis of laser ablation via a scanning galvanometer on a porcine colon tissue model is presented. A thermally damaged region (<85 µm) and fine depth control of ablation using the pulse energies 46 and 33 µJ are demonstrated. It is further demonstrated that such pulses suitable for precision porcine colon resection can be flexibly delivered via a hollow core negative curvature fibre (HC-NCF) and again ablation depth can be controlled with a thermally damaged region <85 µm. Ablation volumes are comparable to that of early stage lesions in the inner lining of the colon. This study concludes that the combination of ultrashort pulses and flexible fibre delivery via HC-NCF present a viable route to new minimally invasive surgical procedures.

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line.

Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.